The phenotypic modulation of vascular smooth muscle cells (VSMCs) from a quiescent, differentiated state to that of proliferating, "undifferentiated cells is a common pathogenic feature of vascular disease. The molecular mechanisms by which this phenotypic "switch" is achieved are unknown. We have observed that proliferating vascular smooth muscle cells express mRNAs that are homologous to a gene expressed in other tissues that acts as a transdominant suppressor of differentiation. This gene is referred to as I (inhibitor of differentiation). It belongs to the family of helix-loop-helix (HLH) family of proteins, many of which are involved in cell determination and differentiation. Using a variety of molecular cloning techniques, we have further identified and cloned four ID-like cDNA from proliferating VSMCs. One of these is the rat homologue of mouse ID1 (i.e. rat ID1) , one is a splicing variant of ID1, one is the rat homologue of ID3, while the fourth one is also a splicing variant of ID3. Unlike the ID1 splicing variant in which the alternative splicing involves the 3' untranslated region, splicing of the ID3 gene occurs in the 2nd helix of th HLH domain, a region critically important for the function of ID. Accordingly, the ID3 variant does not block the ability of basic-HLH proteins, such as myod or E12, to transactivate gene expression from multiple E-boxes. The ID3 variant is not a cloning artefact, but is expressed by tissues with a pattern that is more selective than that of original ID3 mRNA. Expression of rat ID1 is upregulated by a number of growth factors that are important in initiating and sustaining VSMC proliferation, such as PDGF, bFGF, and IGF-1. Expression of rat ID1 is often (but not always) downregulated when VSMCs are made quiescent. However, when cultured VSMCs achieve a level of differentiation comparable to that in the intact vessel (for example, when they are cultured on reconstituted basement membrane), ID expression decreases 10-fold to a leve similar to that seen in the intact, uninjured vessel. Our group is currently initiating studies to identify the function of ID in VSMC migration, proliferation, and differentiation by overexpressing rat ID1 fro constitutively active and inducible promoters, by antisense inhibition of rat ID1, and by genetic screening of cDNA libraries using the two hybrid yeast system to identify potential protein partners for rat ID1.